A variety of diseases are known to be correlated with hyper- or hypophosporylation of proteins. These proteins may be structural proteins or regulatory proteins.
An example of a disease wherein hyperphosporylation of a structural protein is observed is Alzheimer's disease. Here, microtubule-associated tau protein contains unphysiologically high amounts of phosphate residues which may be causative in the formation of paired helical filaments. Further diseases wherein a non-physiological level of phosporylated microtubule-associated proteins is observed include lissencephaly I and Opitz syndrome.
Opitz G/BBB syndrome (MIM 300000) is a congenital disorder that primarily affects the ventral midline. Prominent manifestations include mental retardation associated with dysplasia of the corpus callosum, ocular hypertelorism, cleft lip and palate, tracheo-esophageal fistulas and genitourinary defects. In addition, imperforate anus and hymen and cardiac abnormalities such as tetralogy of Fallot have been described1. The condition is genetically heterogeneous: both an X-linked (Xp22.3) and an autosomal locus (22q11.2; MIM145410) have been described2. The two forms of the disease are clinically indistinguishable. Using a positional cloning approach, we previously identified a candidate gene for the X-linked form, designated MID1, and found that it is selectively mutant in individuals with from OS3.
The protein encoded by MID1 comprises five separate domains common to the RING-finger protein family. A sixth, the C-terminal B30.2 domain, occurs in a subset of these proteins. Most of the mutations identified to date in patients linked to OS cluster in that portion of the MID1 gene. Recently, it was shown that MID1 associates with microtubules, which indicates that it has a physiological role in microtubule dynamics. Mutant forms of MID1 do not associate with microtubules but form cytoplasmic clots instead4.
The N-terminus of MID1 is characterized by a motif (RBCC) consisting of four independent domains: the RING finger, two B-boxes and a coiled-coil domain. This domain structure is conserved throughout the growing family of RING-finger proteins. Formation of macromolecular protein complexes has been described for several of these proteins5,6, whereas heteromeric protein-protein interaction is ascribed to the RBCC motif7. Ubiquitination of target proteins mediated by a RING-finger domain is important in the post-translational regulation of many of proteins8-11.
By yeast two-hybrid screening with MID1 as bait, it is shown that the α4 protein, a regulatory subunit of protein phosphatase 2A (PP2A)12 (Sontag, E.; 2001), interacts with the N-terminal region of MID1. Moreover, it is shown that microtubule-associated PP2A13 is conspicuously upregulated in an embryonic fibroblast cell line derived from an individual with OS. Our data indicate that MID1 is involved in targeting the ubiquitination machinery towards PP2A by binding to its regulatory subunit α4, and that Ser/Thr underphosphorylation of microtubule-associated proteins may be pivotal in the pathogenesis of Opitz syndrome.
Furthermore, it has been shown, that MID1 and MID2 can homo- and heterodimerize and tether α4 to the microtubules, whereby the B-boxes of MID1 and MID2 mediate the interaction with α4 (Short et al.). MID 1 association with microtubules is regulated by dynamic phosphorylation involving MAP kinase and protein phosphatase (Lin et al.).
Whereas the prior art has established that MID1 interacts via α4 with PP2Ac, there has been so far no clue how intracellular amounts of PP2Ac associated with microtubules can effectively be altered. Due to the fact that a number of diseases are correlated with or caused by non-physiological levels of microtubule-associated PP2Ac as mentioned above, there remains a need to provide means and methods of effectively altering these non-physiological intracellular levels of PP2Ac to physiological levels in order to have an effective approach for curing such diseases.
The solution to this technical problem is achieved by providing the embodiments characterized in the claims.